In 1973, a new terminology, non-A, non-B hepatitis (NANB), was introduced to describe a form of hepatitis which was not associated with either the hepatitis A or the hepatitis B viruses. NANB was presumed to be caused by more than one agent. Indeed, two new viruses responsible for the transmission of hepatitis have since been identified. The first one, hepatitis E virus, is transmitted by contaminated water as is the hepatitis A virus. The second one, hepatitis C virus (HCV), similar to the hepatitis B virus, is transmitted mainly through infected blood. Hepatitis C is by far the most common form of viral hepatitis encountered in patients who are seronegative for hepatitis A and B.
Transfusion-associated hepatitis continues to be reported despite massive efforts to identify and eliminate all hepatitis virus infected blood donations. Asymptomatic carriers are common and they frequently progress towards cirrhosis and hepatocellular carcinoma within 10 to 30 years after infection. Depending on the country where it is found, HCV infections account for 50% to 90% of all post-transfusion hepatitis cases and indeed HCV poses a major public health problem worldwide.
Until recently, attempts to identify and isolate HCV were unsuccessful. Two American studies demonstrated that NANB infected sera were often found to have elevated alanine aminotransferase (ALT) activity or were positive for core antigen antibody of the hepatitis B virus (anti-HBc). Accordingly, in 1987, it was decided to screen all blood donations for both ALT and anti-HBc. These tests are believed to have prevented as much as 50% of transfusion-associated NANB hepatitis.
In 1989, HCV was identified and cloned (Choo et al. Science, 244, 349-362, 1989). HCV, a single stranded RNA virus of 10 kb, is a distant relative of the flaviviruses. The sequence of HCV RNA and comparison of that RNA to flavivirus RNA has permitted a general organization of HCV to be proposed. HCV RNA is believed to be read into a single precursor protein which is cleaved into 7 or 8 smaller protein fragments. The first zone of the precursor is believed to include three structural protein molecules called C (core), M (membrane) and E (envelope). However, the M and E proteins might be fused in a single protein. The second zone of the precursor includes five non-structural proteins named NS.sub.1 to NS.sub.5. NS.sub.2 is presumably a protease and NS.sub.5 is probably the RNA polymerase.
Several diagnostic tests for HCV using regions of the HCV viral proteins have been developed (e.g., Kuo et al. Science, 244, 362-364, 1989). One such test uses a protein fragment designated C100-3. It is a fused-protein containing the NS.sub.3 and NS.sub.4 non-structural proteins. In an ELISA this protein has not been shown to be 100% effective. For example, various reports indicate that the test detects only 10%-29% of the clinically diagnosed acute cases of HCV less than 90 days after infection, and only between 67% and 85% of the clinically diagnosed cases of chronic HCV (HCV Learning Guide; Abbott Diagnostics Educational Services, April 1990; Kuo et al. Science, 244, 362-364, 1989; Esteban et al. Lancet 2, 294-296, 1989; van der Poel et al. Lancet 2, 297-298, 1989; Janot et al. Lancet 2, 796-797, 1989; Bruix et al. Lancet 2, 1004-1006, 1989; Colombo et al. Lancet 2, 1006-1008, 1989). Thus, these assays do not display sufficient sensitivity to be useful in a HCV screening program.
The specificity of these HCV assays is also unacceptably low. Some false positive have been attributed to reactions with superoxide dismutase (the HCV C100-3 protein fragment used in the assay is expressed as a fusion protein with superoxide dismutase, SOD) (Ikeda et al. Lancet, 335:1345-6, 1990), to reactions to a rheumatoid factor (Theilman et al. Lancet, 335, 1346, 1990) or to a state of hyperglobulinemia, which is typically associated with autoimmune chronic active hepatitis (McFarlane et al. Lancet, 335, 754-757, 1990; Boudart et al. Lancet, 336, 63, 1990).
In an attempt to improve the sensitivity and specificity of the assays based on C100-3, a recombinant immunoblot assay (RIBA; Chiron-Ortho) has been developed. This test employs two HCV antigens; the recombinant HCV protein C100-3 (which is also called C-100) and the 5-1-1 fragment of C-100 (this gene product covers the C-terminal end of NS.sub.3 and a portion of the N-terminal end of NS.sub.4). SOD is used as a control. The 5-1-1 antigen is produced in E. coli as an SOD fusion. The C-100 peptide is produced in yeast, also as an SOD fusion. Using RIBA to confirm ELISA screening (using C100-3) of a low-risk population, specifically United States blood donors, only 19% of all ELISA repeat positive samples were also found positive by RIBA and 20% were indeterminate (Menitove et al. Lancet, 336, 243-244, 1990).
More recently, another dot-blot assay for HCV was developed by Abbott Laboratories. In this assay, four purified recombinant antigens are spotted on discrete locations on a piece of nitrocellulose paper. One antigen (SOD-C-100) is produced in yeast cells and the other three (C-100, HCV core protein and 33c) are produced in E. coli as fusions with CMP-KDO synthetase (CKS). The 33c antigen includes a large portion of the NS.sub.3 protein. Mimms et al. (Lancet, 336, 1590-1591, 1990) have reported results obtained when this test was used on 153 samples found to be HCV positive by ELISA with C100-3. Only half (75) of the samples were found reactive with the two C-100 gene products--the SOD-C100-3 fusion and the C-100-CKS fusion. The other samples either did not react with any of the four antigens (62) or reacted non-specifically with SOD-C-100 fusion produced in yeast (16).
From the above it can be seen that the recombinant antigen based HCV diagnostic tests now available display poor sensitivity and poor selectivity. They miss a large proportion (15% to 33%) of the chronic cases of HCV and a larger proportion (70% to 90%) of the acute cases. Some 50% of the samples detected are false positives. Furthermore, immunoblot assays are too expensive and laborious for routine screening in blood banks. There is, thus, a definite need to develop a more sensitive and more specific test for the diagnosis of HCV infections.